As the list of expressed human genes expands, a major scientific and medical challenge is to understand the molecular events that drive normal tissue morphogenesis and the progression of pathologic lesions in actual tissue. With refinements in PCR, microhybridization arrays and mutation screening, DNA or mRNA can be extracted from tissue biopsies and analyzed with a parallel panel of hundreds or even thousands of genetic markers. Because cells in complex tissue are biochemically and physically affected by surrounding cells and by remote stimuli from greater distances, the task of analyzing critical gene expression patterns in development, normal function, and disease progression depends on the extraction of specific cells from their complex tissue milieu. Laser capture microdissection (LCM) has been developed by our lab in collaboration with NCI and ORS to provide a rapid, reliable method to procure pure populations of specified cells from specific microscopic regions of tissue sections for subsequent quantitative, multiplex molecular analysis.In the last year we have further refined LCM instrumentation for targeting of single cells and concentrating rare cells onto the transfer surface. We have more fully characterized the thermoplastic polymers we developed for LCM and have used a detailed physical understanding of the activation and capture process to design new approaches including "noncontact" LCM. We have demonstrated reproducible single cell capture and analysis of specific genetic alterations by PCR and sequencing. We are optimizing LCM for quantitative capture and extraction of DNA, protein and mRNA. We have recently made advances in preserving full length mRNA in LCM samples and using this material to generate high quality, complex cDNA libraries of homogeneous populations of cells in developing embryos and in animal models of disease. Through a Collaborative Research and Development Agreement (CRADA) partnership with Arcturus Engineering, Inc (Mountain View, CA), technology advances have been rapidly incorporated in the commercial instruments used in over 400 research labs around the world. LCM-based molecular analysis of histopathological lesions can be applied to any disease process that is accessible through tissue sampling. LCM is being used in the Cancer Genome Anatomy Program (CGAP) effort to catalog the genes which are expressed in human tissue as normal cells undergo premalignant changes and further develop into invasive and metastatic cancer. The fluctuation of expressed genes or alterations in the cellular DNA which correlate with a particular disease stage can be compared within or among individual patients. Such a fingerprint of gene expression patterns may provide crucial clues for etiology, and may ultimately contribute to diagnostic decisions and therapies tailored to the individual patient. Our lab is developing robust statistical tools to be applied to such multidimensional datasets in order to identify critical genes, pathways, and the complex integration of transcription, translation, and post-translational modification of large number of genes that characterize normal cellular function and the deviations characteristic of specific pathologies. Macromolecules found to be uniquely associated with a defined pathological lesion may serve as imaging or therapeutic targets. In animal models, we are currently studying gene expression patterns associated with normal development of the testes and thyroid as well as the induction and progression of plasmacytomas induced by chronic inflammation.